Monoclonal Antibody Drug Development for Alzheimer?s Disease

阿尔茨海默病单克隆抗体药物开发

• 批准号: 7867615
• 负责人: William M Pardridge
• 金额: $ 15.4万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7867615
• 关键词: A Mouse; Affinity; Affinity Chromatography; Age-Months; Alzheimer' s Disease; Alzheimer' s disease model; Amino Acids; Amyloid; Animals; Antibodies; Binding; Binding Sites; Biochemical; Biological Assay; Bioreactors; Blood; Blood - brain barrier anatomy; Blood capillaries; Brain; Brain hemorrhage; Breeding; COS Cells; Cations; Cerebrum; Chimeric Proteins; Chinese Hamster; Chinese Hamster Ovary Cell; Chromatography; Clinical Pharmacology; Clinical Trials; Cloning; Complementary DNA; DNA Sequence; Data; Dementia; Deposition; Disease; Dose; Drug Delivery Systems; Drug Kinetics; Electroporation; Engineering; Fc Receptor; Filtration; G-substrate; Genes; Genetic Engineering; Genotype; Goals; Harvest; Hemorrhage; Histocytochemistry; Human; Hybridomas; IgG1; Immune; Immunoglobulin G; Immunoglobulin Variable Region; Immunoradiometric Assays; Immunotherapy; Inbred BALB C Mice; Injection of therapeutic agent; Insulin Receptor; Label; Laboratories; Light; Measurement; Mediating; Mediation; Medicine; Methods; Monoclonal Antibodies; Mus; Ovary; Peptides; Peripheral; Peritoneal; Pharmaceutical Preparations; Plasma; Plasmids; Proteins; Prussian blue; Public Health; Publishing; RNA Splicing; Radio; Rattus; Reaction; Research; Rodent; Saline; Senile Plaques; Series; Serum-Free Culture Media; Staging; Structure; Testing; Therapeutic; Transfection; Transferrin Receptor; Transgenic Mice; Validation; Western Blotting; Work; abstracting; amyloid peptide; animal efficacy; antibiotic G 418; capillary; drug development; drug discovery; human INSR protein; in vivo; interest; lipofection; molecular trojan horse; mouse model; nervous system disorder; neurobehavior; novel therapeutics; pre-clinical; receptor; receptor binding; uptake; variable region gene

ABSTRACT The dementia of Alzheimer's disease (AD) is caused by the deposition of A¿ amyloid in brain over many years. Once the amyloid plaque forms in brain, it is permanent in the absence of plaque disaggregation therapy. The most potent form of plaque diasaggregation therapy is a monoclonal antibody (MAb) against the A¿ amyloid peptide of AD, and passive immune therapy of AD is currently being tested in clinical trials. However, the anti- A¿ MAb does not cross the blood-brain barrier (BBB). Consequently, very high doses of MAb must be administered to lower plaque in brain of AD transgenic mice, and these high doses causes brain microhemorrhage. The present research will genetically engineer a new form of anti-A¿ MAb that is enabled to cross the BBB via receptor- mediated transport in both the blood-to-brain and brain-to-blood directions. The PI has previously genetically engineered a chimeric MAb against the mouse transferrin receptor (TfR) that crosses the BBB in the blood-to-brain direction via receptor-mediated transport on the mouse BBB TfR, and also crosses the BBB in the brain-to-blood direction on the BBB Fc receptor (FcR). In addition, the PI has genetically engineered, and expressed a single chain Fv (ScFv) antibody against the amino terminal portion of the A¿ peptide. The present research will produce a new fusion protein, wherein the anti-A¿ ScFv is fused to the carboxyl terminus of the chimeric MAb against the mouse TfR, and this new fusion protein is designated the TfRMAb-A¿ScFv bi-specific antibody (BSA). Following the genetic engineering of the new BSA, the protein will be transiently expressed in COS cells, and the bi- functionality of the BSA will be demonstrated with a mouse TfR binding assay and an A¿ binding assay. The BSA will then be permanently expressed in Chinese hamster ovary (CHO) cells, followed by selection, and dilutional cloning, and purification with affinity and cation exchange chromatography. The purified BSA will then be used to treat APPswe/PSEN1(dE9) double transgenic mice over 3 month period. Control mice will be treated with either saline or with the conventional high dose anti-A¿ MAb that does not cross the BBB. The mice will be evaluated for neurobehavior, plasma A¿ levels, brain A¿ plaque content, and brain micro-hemorrhage using Prussian blue histochemistry. This research will provide the necessary pre-clinical pharmacology to support an IND filing for the treatment of humans with AD using genetically engineered fusion antibodies that both cross the BBB via receptor-mediation and bind and disaggregate A¿ amyloid plaque of AD. RELEVANCE TO PUBLIC HEALTH The dementia of Alzheimer's disease (AD) is caused by the deposition of A¿ amyloid in brain over many years, and once the amyloid plaque forms in brain, it is permanent in the absence of plaque disaggregation therapy. The most potent form of plaque diasaggregation therapy is a monoclonal antibody (MAb) against the A¿ amyloid peptide of AD, and passive immune therapy of AD is currently being tested in clinical trials; however, the anti- A¿ MAb does not cross the blood-brain barrier (BBB). The present research will test in AD transgenic mice the efficacy of a new genetically engineered fusion antibody that both crosses the BBB via receptor-mediation and binds and disaggregates A¿ amyloid plaque of AD.

摘要 阿尔茨海默病（Alzheimer's disease，AD）是由于A β淀粉样蛋白在脑内过度沉积而引起的痴呆。 多年一旦淀粉样斑块在大脑中形成，在没有斑块的情况下是永久性的 分解疗法斑块解聚疗法的最有效形式是单克隆抗体， 抗AD淀粉样肽的抗体（MAb），目前AD的被动免疫治疗 正在进行临床试验然而，抗A单克隆抗体不能穿过血脑屏障（BBB）。 因此，必须给予很高剂量的MAb以降低AD脑内斑块 转基因小鼠，这些高剂量会导致脑微出血。本研究将 基因工程设计一种新形式的抗A单克隆抗体，使其能够通过受体- 介导的运输在血液到脑和脑到血液的方向。此前，PI 基因工程化的嵌合单克隆抗体抗小鼠转铁蛋白受体（TfR），该受体与 通过小鼠BBB TfR上受体介导的转运，在血脑方向上对BBB进行转运， 在BBB Fc受体（FcR）上以脑-血方向穿过BBB。此外，PI 基因工程，并表达针对氨基末端的单链Fv（ScFv）抗体， A肽的一部分。本研究将产生一种新的融合蛋白，其中抗A？ ScFv融合到针对小鼠TfR的嵌合MAb的羧基末端，并且这种新的 融合蛋白被命名为TfRMAb-A？ScFv双特异性抗体（BSA）。随着基因 新BSA的工程化，蛋白质将在COS细胞中瞬时表达，并且双- BSA的功能将通过小鼠TfR结合试验和A？结合试验来证明。 比色法然后BSA将在中国仓鼠卵巢（CHO）细胞中永久表达， 通过选择和稀释克隆，以及用亲和和阳离子交换纯化 层析然后将纯化的BSA用于处理APPswe/PSEN 1（dE 9）双转基因小鼠。 小鼠在3个月内。对照小鼠将用生理盐水或常规高浓度的生理盐水处理。 剂量的抗A单克隆抗体不能穿过血脑屏障。将评估小鼠的神经行为， 血浆A <$水平、脑A <$斑块含量和使用普鲁士蓝的脑微出血 组织化学本研究将提供必要的临床前药理学，以支持IND 申请使用基因工程融合抗体治疗AD患者， 通过受体介导穿过血脑屏障，结合并分解AD的A？淀粉样斑块。与公共卫生的关系 阿尔茨海默氏病（AD）的痴呆是由A？淀粉样蛋白在大脑中的沉积引起的。 大脑中的淀粉样蛋白斑块一旦形成，它是永久性的， 缺乏斑块解聚治疗。最有效的斑块形式 解聚疗法是一种抗A淀粉样肽的单克隆抗体 AD的被动免疫治疗目前正在临床试验中进行测试; 然而，抗A单抗不穿过血脑屏障（BBB）。本 研究将在AD转基因小鼠中测试一种新的基因工程药物的功效。 融合抗体，其通过受体介导穿过BBB并结合， 分解AD的淀粉样斑块。